JP6073857B2 - Synthetic antimicrobial effect - Google Patents

Description

  The present invention relates to the field of biocontrol, and in particular, to an antimicrobial composition comprising a Lactobacillus rhamnosus strain and / or a Lactobacillus paracasei strain. Furthermore, the present invention uses such antimicrobial compositions to produce foods, feeds and pharmaceutical products containing such antimicrobial compositions, to produce such foods, feeds and pharmaceutical products. Methods and methods for reducing the content of unwanted microorganisms in such food, feed and pharmaceutical products.

  Bioprotective cultures are used in various food applications as an alternative to chemical preservatives. In dairy products, contamination with yeast and mold can limit the shelf life of the dairy product. A product area that experiences significant problems of yeast and mold contamination includes fresh cheese.

  Bioprotective solutions to solve these problems have been developed in the dairy industry. The most famous and perhaps the most commonly used antifungal bioprotection solutions on the market are HOLDBAC ™ YM-b and HOLDBAC ™ YM-C from Danisco, both of which are Propionibacterium And a combination of Lactobacillus subspecies.

  The use of propionic acid bacteria together with lactic acid bacteria for the inhibition of microorganisms is disclosed in patent applications US 2005/0095318 and WO 2004/041305.

  Propionibacterium subsp. Is believed to contribute to antifungal activity primarily through the production of acetic acid and other metabolites in addition to the production of propionic acid.

  However, these species are quite expensive to produce on an industrial scale, and the production of propionic acid can cause alterations that are unacceptable to the final dairy product.

  Other commercially available antifungal biodefense solutions for dairy products include Lyofast LPRA from Sacco and Aroma-Prox® RP80 from BIOPROX. Both of these products contain a combination of Lactobacillus plantarum and Lactobacillus rhamnosus. Tharmaraj and Shah (2009) describe the screening of bioprotective lactic acid bacteria candidates in cheese-based dip. Lactobacillus rhamnosus and Lactobacillus paracasei tested as single strains showed the greatest inhibitory effect on yeast and mold. However, the combination of Lactobacillus rhamnosus and Lactobacillus paracasei was not tested.

  Therefore, there is industrial acceptance in developing improved biodefense solutions that can refrain from using Propionibacterium spp. Without compromising antifungal effects.

  The present invention has identified a new strain of lactic acid bacteria that is markedly effective in inhibiting the growth of bacterial and fungal microorganisms known as contaminants in milk and dairy products. Furthermore, the present invention has found that certain lactic acid bacteria exhibit a significant synergistic antimicrobial effect when combined with other types of lactic acid bacteria. The antimicrobial effect of the two types of bacteria combined is surprisingly greater than the combined effect of using the two types of bacteria alone.

  Accordingly, a first aspect of the present invention relates to an antimicrobial composition comprising one or more Lactobacillus rhamnosus strains and one or more Lactobacillus paracasei strains. In a preferred embodiment, the antimicrobial composition comprises one or more selected from the group consisting of (a) Lactobacillus rhamnosus CHCC12697 and Lactobacillus rhamnosus CHCC14226 and mutants thereof. One or more Lactobacillus paracasei strains selected from the group consisting of Lactobacillus rhamnosus strains, and (b) Lactobacillus paracasei CHCC12777 and mutants thereof. To do. In another preferred aspect, the present invention relates to a Lactobacillus rhamnosus CHCC12697, Lactobacillus rhamnosus CHCC14226, a Lactobacillus paraCC27 mutant strain CC1 An antimicrobial composition containing one or more strains is provided, wherein the mutant strain is obtained using the strain deposited as starting material.

  The composition of the present invention provides the advantage that it can inhibit the growth of unwanted microorganisms selected from fungi, bacteria and mixtures thereof in human and animal food, feed and pharmaceutical products and the like. The prevention and / or inhibition of the growth of fungi such as yeasts and molds is particularly envisaged. Thus, in a preferred embodiment, the term “antimicrobial” should be understood as “antifungal”. Accordingly, a second aspect of the invention relates to the use of the antimicrobial composition of the first aspect in the preparation of food, feed and pharmaceutical products.

  The third aspect relates to the use of the antimicrobial composition of the first aspect for inhibiting the growth of unwanted microorganisms selected from the group consisting of fungi, bacteria and mixtures thereof. In particular, the compositions of the present invention can be used to inhibit and / or prevent the growth of fungi and bacteria, which are known contaminants in dairy processes, such as the milk fermentation process.

  The fourth aspect relates to the use of the antimicrobial composition of the first aspect as a pharmaceutical product. The pharmaceutical product is preferably used to treat patients infected with bacteria or fungi, preferably mold.

  The fifth aspect relates to a food, feed or pharmaceutical product containing the antimicrobial composition of the first aspect of the present invention.

A sixth aspect of the present invention relates to a method for producing a food, feed or pharmaceutical product according to the fifth aspect of the present invention, and the method comprises the steps of the present invention in the course of producing the food, feed or pharmaceutical product. Adding an antimicrobial composition of one aspect. Wherein the method comprises the addition of an antimicrobial composition comprising a combination of a Lactobacillus rhamnosus strain and a Lactobacillus paracasei strain, the composition comprising one or more lactobacillus strains. Bacillus rhamnosus (Lactobacillus rhamnosus) strains and one or more Lactobacillus paracasei (Lactobacillus paracasei) strain, the food for feed or pharmaceutical product, each 1x10 ⁶ cfu / g, preferably at least 5x10 ⁶ cfu / g above, or respectively 1x10 ⁶ cfu / ml or more, preferably to 5x10 ⁶ cfu / ml or more, or the food, feed or pharmaceutical product surface, 1x10 ⁵ fu / cm ^2, is preferably added at a concentration of 1x10 ⁷ cfu / cm ^2. In the course of the production, the production parameters are such that the concentration of one or more Lactobacillus rhamnosus strains and one or more Lactobacillus paracasei strains increases or becomes constant. To be controlled.

  In a seventh aspect, the present invention relates to Lactobacillus rhamnosus CHCC12697, or a mutant thereof, deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM24616. , Obtained using the stock deposited as starting material.

In an eighth aspect, the present invention relates to Lactobacillus rhamnosus CHCC14226, or a mutant strain thereof, deposited under the accession number DSM24652 to German Collection of Microorganisms and Cell Cultures (DSMZ). , Obtained using the stock deposited as starting material.
Lactobacillus paracasei CHCC12777 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM24651;

  In a ninth aspect, the present invention relates to a Lactobacillus paracasei CHCC12777 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM24651, or a mutant strain thereof. , Obtained using the stock deposited as starting material.

FIG. 1 shows that the starting culture alone (Reference, column 1), Lb. Rb. Rhamnosus CHCC12697 (column 2), Lb. Paracasei CHCC 12777 (column 3), or Lb. Rb. Rhamnosus CHCC12697 and Lb. FIG. 5 shows yeast and mold growth on plates prepared from milk fermented with a combination of Lb. paracasei CHCC12777 (column 4). In the top plate, from the left: K. marxianus, P.M. Fermentans, P. fermentans, Y.M. Y. lipolytica and C.I. Target contaminants of salmon (C. sake) were added at the concentrations indicated in the text. In the second stage plate, P.I. P. nalgiovense is at the top, Cladiosporium spp. Is in the lower left, and P. A commune was added at the lower right. In the last plate, Mucor ssp. Was added alone. These plates were incubated at 7 ± 1 ° C. for 17 days.

FIG. 2 shows CHN-19 as a starting culture alone (black diamond shape) or the following strains: HOLDBAC ™ YM-B (white square), HOLDBAC ™ YM-C (white diamond), Lb . Parabasei CHCC12777 (black square), Lb. Rhamnosus CHCC12697 (black triangle), or Lb. Paracasei CHCC12777 and Lb. FIG. 5 shows cell counts of Yarrowia lipolytica isolates added to fatty milk inoculated with a combination of Lb. rhamnosus CHCC12697. All were added prior to fermentation at 29 ± 1 ° C. until pH 4.65 ± 0.05 was reached.

FIG. 3 shows that the starting culture alone (Reference, column 1), Lb. Rb. Rhamnosus CHCC 14226 (column 2), Lb. Parabasei CHCC12777 (column 3), or Lb. Rb. Rhamnosus CHCC14226 and Lb. FIG. 4 shows yeast and mold growth on plates prepared from milk fermented with a combination of Lb. paracasei CHCC12777 (column 4). From the left in the upper plate: K. marxianus, P.M. Fermentans, P. fermentans, Y.M. Y. lipolytica and C.I. Target contaminants of salmon (C. sake) were added at the concentrations indicated in the text. In the lower plate, P.I. P. nalgiovense is at the top, Claudiosporium spp. Is in the lower left, and P. n. A commune was added at the lower right. These plates were incubated at 7 ± 1 ° C. for 17 days.

FIG. 4 shows that CHN-19 alone (black diamond shape) or the following strains: HOLDBAC ™ YM-B (white square), HOLDBAC ™ YM-C (white diamond), Lb . Parabasei CHCC12777 (black square), Lb. Rhamnosus CHCC14226 (black triangle), or Lb. Paracasei CHCC12777 and Lb. FIG. 6 shows cell counts of Klyveromyces marxianus isolates added to fatty milk inoculated with a combination of Lb. rhamnosus CHCC14226. All were added prior to fermentation at 29 ± 1 ° C. until pH 4.65 ± 0.05 was reached.

FIG. 5 shows that the starting culture alone (Reference, first photo), Lb. Paracasei CHCC14676 (second photo), Lb. Rhamnosus CHCC5366 (third photo), or Lb. Paracasei CHCC 14676 and Lb. Figure 3 shows the growth of mold on plates prepared from milk fermented with a combination of Lb. rhamnosus CHCC5366 (column 4). From upper left to lower right: Penicillium nargiovese, Penicillium commune, Aspergillus versicolor, and Penicillium versicum, Were added at the concentrations indicated. These plates were incubated at 7 ± 1 ° C. for 12 days.

FIG. 6 shows that the starting culture alone (Reference, first photograph), Lb. Paracasei CHCC14676 (second photo), Lb. Rhamnosus CHCC14226 (3rd photo), or Lb. Paracasei CHCC 14676 and Lb. Figure 4 shows the growth of mold on plates prepared from milk fermented with a combination of Lb. rhamnosus CHCC 14226 (column 4). From upper left to lower right: Penicillium nargiovese, Penicillium commune, Aspergillus versicolor, and Penicillium versicum, Were added at the concentrations indicated. These plates were incubated at 7 ± 1 ° C. for 12 days.

FIG. 7 shows that the starting culture alone (Reference, first photograph), Lb. Paracasei CHCC12777 (second photo), Lb. Rhamnosus CHCC14226 (3rd photo), or Lb. Paracasei CHCC12777 and Lb. Figure 4 shows the growth of mold on plates prepared from milk fermented with a combination of Lb. rhamnosus CHCC 14226 (column 4). From upper left to lower right: Penicillium nargiovese, Penicillium commune, Aspergillus versicolor, and Penicillium versicum, Were added at the concentrations indicated. These plates were incubated at 7 ± 1 ° C. for 12 days.

FIG. 8 shows the experimental results of sour cream to which Lactobacillus paracasei CHCC12777 was added. P. Commune (P. commune) (M6), A.I. A. versiccolor (M7), P.M. P. brevicompactum (M1), P. brevicompactum. P. crustosum (M10) and P. crustosum P. glabrum (M8) is active on sour cream produced from milk fermented with the starting culture alone (upper) or with the starting culture and the HOLDBAC ™ YM-B culture (middle). Proliferated. In contrast, when Lactobacillus paracasei CHCC12777 was present during fermentation of milk (bottom), the growth of all molds tested was inhibited.

FIG. 9 shows the experimental results of a tobolog with the addition of Lactobacillus rhamnosus CHCC12697. Mucor ssp. Was shown to proliferate actively on tovalogs produced from milk fermented with only the starting culture strain CHN-19 (Ref.). The mold grew actively on tobalog produced from milk fermented with the starting culture and the HOLDBAC ™ YM-B culture, but fermented with the starting culture and the HOLDBAC ™ YM-C culture. It did not grow much on Tobalog produced from milk. However, when Lactobacillus rhamnosus strain CHCC12697 was present during fermentation of milk (bottom), significant inhibition of Mucor ssp. Growth was observed.

FIG. 10 shows the experimental results of yogurt added with Lactobacillus rhamnosus CHCC14226. On yogurt produced from milk fermented with starting culture YF-L901 alone (upper) or starting culture and HOLDBAC ™ YM-B (middle). P. brevicompactum (M1), P. brevicompactum. Commune (P. commune) (M6), A.I. A. versicolor (M7) and P.I. P. crussum (M10) proliferated actively. In contrast, when Lactobacillus rhamnosus CHCC14226 was present during fermentation of milk (bottom), growth of any of the tested molds was inhibited.

FIG. 11 shows K. in Tobalog. 1 shows a quantitative determination of the inhibitory effect of Lactobacillus rhamnosus CHCC12777 and Lactobacillus rhamnosus CHCC12697 on K. lactis. In the presence of Lactobacillus rhamnosus CHCC12777 with starting culture CHN-19 and Lactobacillus rhamnosus CHCC12697 in the presence of starting culture CHN-19 prior to fermentation, It was demonstrated that the growth of K. lactis was inhibited. Both Lactobacillus paracasei CHCC12777 and Lactobacillus rhamnosus CHCC12697 caused significantly higher inhibition than the commercial culture HOLDBAC ™ YM-B.

FIG. 12 shows a quantitative determination of the inhibitory effect of Lactobacillus rhamnosus CHCC14226 against Debaromyces hansenii in yogurt. If Lactobacillus rhamnosus CHCC14226 is present with the starting culture YF-L901 prior to fermentation, It has been demonstrated that the growth of D. hansenii is inhibited. This strain caused significantly higher inhibition than the commercially available culture HOLDBAC ™ YM-B.

Definitions As used herein, “lactic acid bacteria” refers to gram-positive microaerobic or anaerobic bacteria that produce acids containing lactic acid as the primary acid produced in sugar fermentation. The most useful lactic acid bacteria in the industry include Lactococcus spp., Streptococcus spp., Lactobacillus spp., Leuconostoc spp. “Lactobacilli spp.”, “Lactobacilli spp.”, “Lactobacilli spp.”, “Lactobacilli spp.”, “Lactobacillus sp.” Belongs. These are often used as food cultures alone or in combination with other lactic acid bacteria.

  Lactic acid bacteria, including bacteria of the species Lactobacillus sp. And Streptococcus thermophilus, are commonly used in the dairy industry as frozen or lyophilized cultures for bulk starter seedlings. Alternatively, it is supplied as a so-called Direct Vat Set (DVS) culture intended to be seeded directly on fermenting vessels or bats for the production of fermenters or dairy products such as cheese. Such lactic acid bacteria cultures are commonly referred to as “starter cultures” or “starter strains”.

  As used herein, the term “mesophile” refers to the most proliferating microorganisms at moderate temperatures (15 ° C. to 40 ° C.). The most useful mesophilic bacteria in the industry include Lactococcus spp. And Leuconostoc spp. As used herein, the term “medium temperature fermentation” refers to fermentation at a temperature between about 22 ° C. and about 35 ° C. The term “mesophilic dairy product” refers to a fermented dairy product prepared by mesophilic fermentation of a mesophilic starting culture, such as buttermilk, sour milk, cultured milk, smetana, sour cream and Fresh cheeses such as Quark, Tobalog and cream cheese can be mentioned.

  As used herein, the term “thermophilic” refers to the most proliferating microorganisms at temperatures above 43 ° C. The most useful thermophilic bacteria in the industry include Streptococcus spp. And Lactobacillus spp. As used herein, the term “thermophilic fermentation” refers to fermentation at temperatures above about 35 ° C., such as at about 35 ° C. to about 45 ° C. The term “thermophilic dairy product” refers to a fermented dairy product prepared by thermophilic fermentation of a thermophilic starting culture, such fermented dairy products include set yogurt, starred yogurt and drinking yogurt.

  The term “milk” should be understood as a milk secretion obtained by milking any mammal, such as cows, sheep, goats, buffalo or camels. In a preferred embodiment, the milk is milk. Milk also includes protein / fat solutions produced from plant material, such as inputs.

  The term “dairy substrate” refers to any raw / dairy material that can be subjected to fermentation according to the method of the present invention. Thus, useful dairy substrates include, but are not limited to, any milk or milk-like product solution / suspension including protein, such as whole or low fat milk, skim milk, butter milk, reconstituted milk powder, condensed milk, Contains dry milk, whey, whey permeate, lactose, mother liquor from lactose crystallization, whey protein concentrate, or cream. Obviously, the dairy substrate may be from any mammal, such as substantially pure mammalian milk, or reconstituted milk powder. Prior to fermentation, the dairy substrate may be homogenized or sterilized according to methods known in the art.

  As used herein, the term “homogenization” means vigorous mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it should be performed so that the milk fat is dispersed to such a small size that it does not separate from the milk. This can be accomplished by applying high pressure to the milk with a small orifice.

  As used herein, “sterilizing” means treating a dairy substrate to reduce or kill live organisms, such as microorganisms. Preferably, sterilization is achieved by maintaining the milk substrate at a specific temperature for a period of time. The specific temperature is usually achieved by heating. The sterilization temperature and time can be selected to kill or inactivate specific bacteria, such as harmful bacteria. A rapid cooling process may follow.

  “Fermentation” in the present invention means that carbohydrates are converted into alcohols or acids by the action of microorganisms. Preferably, the method of the present invention comprises converting lactose to lactic acid.

  Fermentation processes used to produce dairy products are well known and the skilled person knows how to select appropriate processing conditions such as temperature, oxygen content, microbial content and characteristics, and processing time. . Obviously, the fermentation conditions are selected to support the achievement of the present invention, ie to obtain a dairy product that is solid (such as cheese) or liquid (such as a fermented dairy product).

  As used herein, “unwanted microorganism” refers to microorganisms such as bacteria and fungi, such as yeast, that are pathogenic and / or degrade food, feed or pharmaceutical products.

  The terms “inhibit” and “to inhibit” with respect to unwanted microorganisms refer to, for example, the number or density of unwanted microorganisms in a food product and / or on the surface of a food product, or with the addition of an antimicrobial composition. It means that the growth rate is less in the food product without such antimicrobial composition and / or on the food product surface.

  In the present specification, the term “mutation” is to be understood as a strain derived from the strain of the present invention, produced by means such as genetic manipulation, radiation and / or chemical treatment. Mutations are functionally equivalent mutations, such as having substantially the same or improved characteristics compared to the parent strain (eg, diacetyl production, viscosity, gel strength, mouth coating, flavor, post Acidification, rate of acidification and / or phage robustness mutations. Such mutations are part of the present invention. In particular, the term “mutation” is commonly used, including treatment with chemical mutagens such as ethanemethanesulfonic acid (EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UV light and the like. Strains obtained by subjecting the strains of the present invention to any mutagenesis treatment, or spontaneously occurring mutations. Mutations may have been subjected to multiple mutagenesis processes (one process should be understood as a mutagenesis process followed by a screening / selection step), but preferably no more than 20, or Desirably, no more than 10 or 5 treatments (or screening / selection steps) are performed. In preferred mutations, less than 5%, or less than 1%, or less than 0.1% of the nucleotides in the bacterial genome are changed to another nucleotide or deleted compared to the parent strain.

  In the description of the present application, the terms “subject”, “above”, “one”, and similar terms thereof (particularly the claims) are singular unless otherwise stated or explicitly denied. And a plurality are envisioned. The terms “including”, “having”, “containing”, and “including” are understood as non-limiting terms (ie, including but not limited to) unless otherwise stated. Should. The recitation of numerical ranges herein is intended to be considered merely a simple way to individually reference each individual numerical value within that range, unless otherwise stated. Are hereby incorporated by reference as if individually described herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted. Any and all example embodiments or exemplary terms (such as “for example”) in this specification are intended only to better illustrate the present invention and, unless stated otherwise, It does not limit the range. No language in the specification should be construed as indicating any non-specified element essential to the practice of the invention.

Implementation and Aspects of the Invention The inventors have eagerly provided an antimicrobial composition containing one or a mixture of two different lactic acid strains effective against unwanted microorganisms such as mold and yeast. Screening and research were conducted.

  The present invention provides 200 strains of Lactobacillus plantarum, Lactobacillus paracasei to find the most effective single or combination strains against a wide range of microorganisms such as yeast and mold. And candidates for Lactobacillus rhamnosus were screened. Screening is performed in a model assay using a milk-based medium that mimics the mesophilic fermented dairy product as much as possible, to which the starting culture is added, along with a bioprotectant candidate or alone, and It was fermented under the conditions employed in mesophilic fermented dairy products. Target microorganisms were isolated from mesophilic fermented dairy products. Lactic acid bacteria isolated from HOLDBAC ™ culture strain provided by Danisco A / S, Denmark, and complete HOLDBAC ™ YM-B and HOLDBAC ™ YM-C containing both lactic acid bacteria and propionic acid bacteria Used as a benchmark.

  When 17 candidates out of Lactobacillus paracasei and Lactobacillus rhamnosus were tested at 25 ° C., the equivalent of, or more prominently, the 12 indicators Inhibited mold. Accordingly, in a first aspect, the present invention relates to an antimicrobial composition comprising one or more strains of Lactobacillus paracasei or Lactobacillus rhamnosus.

  The nine identified candidate strains also showed similar activity as the benchmark lactic acid bacteria when tested under low temperature conditions, in particular, Lactobacillus paracasei CHCC12777 with accession number DSM 24651, Lactobacillus with accession number DSM24616. Three strains of Lactobacillus rhamnosus CHCC12597 and accession number DSM24652 Lactobacillus rhamnosus CHCC14226 have been shown to be very effective. These three strains were deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ).

These strains, when tested in a so-called bottle assay, exhibited a significant or significant effect on a single, different yeast compared to the HOLDBAC ™ culture. These single strains are therefore very suitable for use as antimicrobial agents. In preferred embodiments, the present invention provides the following groups:
Lactobacillus rhamnosus CHCC12697, deposited with German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number DSM24616,
Lactobacillus rhamnosus CHCC 14226, deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM24652,
Lactobacillus paracasei CHCC12777 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM24651;
And one or more strains selected from those mutant strains, wherein the mutant strains are obtained using said strains deposited as starting material .

  In a particularly preferred embodiment, the antimicrobial composition of the present invention contains the above strain or one of their mutations as one agent having antimicrobial activity.

  Apart from the above three strains, the present invention also contemplates mutations derived from these strains, ie mutant strains obtained using one of the CHCC12777, CHCC12697 or CHCC14226 strains deposited as starting material. . Mutant strains may be made from the starting materials using, for example, genetic engineering, irradiation, UV irradiation, chemical treatment, and / or any method that induces changes in the genome. The mutations of the present invention may inhibit or prevent the growth of certain bacteria or fungi, preferably molds. The mutation is, for example, yeast K. as a contaminant. marxianus, P.M. fermentas, Y.M. lipolytica or C.I. 80% or more, 90% or more, 95% or more, or 100% or more of an antimicrobial, such as antifungal, as determined by the assay described in Example 2 using one of the sake Has activity.

  The inhibitory effect of these strains on unwanted microorganisms can be determined by storing the bottles at an appropriate temperature for an appropriate storage period, as described in the examples below.

  In general, the appropriate temperature at which this method is to be performed depends on the temperature at which the particular food, feed or pharmaceutical product is usually stored and / or manufactured. The temperature at which the product is normally stored is 5 ° C to 26 ° C, preferably about 8 ° C.

  The storage time at the temperature depends on the period (shelf life) in which the food, feed or pharmaceutical product is normally stored. The normal storage period is 7 to 28 days, preferably about 21 days.

  When testing a combination of two strains, the most effective single strain combination is the same when the total concentration of added cells is tested in a single test and in a combination of two strains. Even so, it was better than using those strains alone. Lactobacillus paracasei CHCC12777 and Lactobacillus rhamnosus CCC12697 or Lactobacillus paracasei Lactobacillus Lactobacillus Was more effective than HOLDBAC ™ YM-B and HOLDBAC ™ YM-C from Danisco, Denmark.

  Accordingly, in a preferred embodiment, the present invention relates to an antimicrobial composition comprising one or more Lactobacillus rhamnosus and one or more Lactobacillus paracasei. Lactobacillus rhamnosus is Lactobacillus rhamnosus with accession number DSM24616 (Lactobacillus rhamnosus), Lactobacillus rmunosus with accession number DSM24652, Lactobacillus rhamnosus The mutant strain is deposited as a starting material. Acquired using the above stock.

  In another preferred embodiment, the one or more Lactobacillus paracasei is selected from the group consisting of Lactobacillus paracasei with accession number DSM 24651 CHCC12777 and mutants thereof, wherein the mutation A strain is obtained using said strain deposited as starting material.

In a further aspect, the present invention relates to an antimicrobial composition, more preferably an antifungal composition, comprising one or more Lactobacillus rhamnosus and one or more Lactobacillus paracasei. ,here,
The one or more Lactobacillus rhamnosus is Lactobacillus rhamnosus with accession number DSM 24616 CHCC12697, Lactobacillus rmusnosil with the accession number DSM24652 Wherein the mutant strain is obtained using the above strain deposited as starting material, and the one or more Lactobacillus paracasei is a Lactobacillus acc. Lactobacillus paracasei CHCC12777 and It is selected from the group consisting of mutant, wherein said mutant strain is obtained using the strains deposited as a starting material.

  Particularly preferred is an antimicrobial composition containing one or more Lactobacillus rhamnosus CHCC12697 and one or more Lactobacillus paracasei CHCC12777. Similarly, antimicrobial compositions containing one or more Lactobacillus rhamnosus CHCC14226 and one or more Lactobacillus paracasei CHCC12777 are also particularly preferred in the present invention.

Typically, the antimicrobial composition contains bacteria in a concentrated form, such as a frozen, dried, or lyophilized concentrate, and typically the concentration of viable cells is between 10 ^{4 and} 10 per gram of composition. ¹² cfu (colony forming unit), for example, 10 ⁴ cfu / g or more, such as 10 ⁵ cfu / g or more, such as 10 ⁶ cfu / g or more, such as 10 ⁷ cfu / g or more, such as 10 ⁸ cfu / g or more, For example, it is 10 ⁹ cfu / g or more, for example, 10 ¹⁰ cfu / g or more, for example, 10 ¹¹ cfu / g or more. Accordingly, the antimicrobial composition of the present invention is preferably present in frozen, dried or lyophilized form, for example as a Direct Vat Set (DVS) culture. However, as used herein, an antimicrobial composition is a liquid obtained by suspending the frozen, dried or lyophilized cell concentrate in a liquid medium such as water or PBS. May be. When the antimicrobial composition of the present invention is a suspension, the concentration of viable cells is 10 ⁴ to 10 ¹² cfu (colony forming unit) per ml of the composition, for example, 10 ⁴ cfu / ml or more, for example 10 ^5. cfu / ml or more, such as 10 ⁶ cfu / ml or more, such as 10 ⁷ cfu / ml or more, such as 10 ⁸ cfu / ml or more, such as 10 ⁹ cfu / ml or more, such as 10 ¹⁰ cfu / ml or more, such as 10 ¹¹ cfu / ml. More than ml.

  The composition further comprises, as further components, cryoprotectants and / or known additives such as yeast extract, sugars and vitamins such as vitamins A, C, D, K or vitamin B vitamins You may contain. Suitable cryoprotectants that can be added to the compositions of the present invention are refractory to microorganisms such as mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose and fructose. It is an ingredient that improves. Other additives include carbohydrates, perfumes, minerals, enzymes (eg rennet, lactase and / or phospholipase).

  In an antimicrobial composition of the present invention comprising a Lactobacillus rhamnosus strain and a Lactobacillus paracasei strain, the Lactobacillus strain Lactobacillus rhamnoslus Lactobacillus strain The ratio of, for example, the concentration or number of Lactobacillus rhamnosus and Lactobacillus paracasei strains is preferably 1: 100 to 100: 1, preferably 1:10 to 10: 1. It is.

  The antimicrobial compositions of the present invention can be used in any food, feed and pharmaceutical product that is susceptible to microbial degradation and / or contamination with yeast and mold. These include, but are not limited to, fruits and vegetables including dried products, cereal and cereal derived products, dairy products, animal meat, poultry and marine products. In a particularly preferred embodiment, the composition is used for dairy products and / or animal and poultry. In a preferred embodiment, the composition of the invention is used as an additive in the preparation of dairy products such as yogurt, tobalog, sour cream, cream cheese and the like.

  The antibacterial compositions of the present invention can also be used to inhibit unwanted microorganisms selected from the group consisting of fungi and bacteria and mixtures thereof. The compositions of the present invention are particularly useful for inhibiting and / or preventing the growth of fungi and bacteria, which are common contaminants in dairy processes such as the milk fermentation process.

  In a particularly preferred embodiment, the antimicrobial composition of the present invention is used against fungi such as yeast and mold. This means that the composition is used to inhibit and / or prevent fungal growth which causes contamination in dairy processes, in particular milk fermentation processes. The antimicrobial compositions of the present invention may be used in yeasts such as Klyveromyces (eg K. marxianus, K. lactis), Pichia (eg P. (fermentans)). fermentans), Yarrowia (eg, Y. lipolytica), Candida (eg, C. sake), and other yeasts; or molds, eg, Penicillium (eg, P. pylori). Nalgiovense, P. commune, P. crustosum, P. brevicompactum, P. glabra (Glabrum), Mucor sop., Cladiosporium ssp., Aspergillus (eg A. versicolor), Debaromikes (eg. Debaromyces. It can be used to inhibit and / or prevent the growth of molds of the genus such as Hansenii, Klyveromyces marxianus, Yarrowia lipolytica, Penicillium p. ), Cladosporium ssp., Peni Penicillium commune, Mucor ssp., Penicillium brevicompactum, Aspergillus versicolor, Penicillium cus It is particularly preferred to use the antimicrobial composition of the invention for the inhibition and / or prevention of the growth of Lactis (Kluyveromyces lactis) and / or Debaromyces hansenii.

  The antimicrobial composition of the first aspect of the invention may be used as a pharmaceutical composition for treating infections of pathogenic fungi, preferably pathogenic yeasts.

  As mentioned above, one aspect of the present invention relates to a food, feed or pharmaceutical product containing the antimicrobial composition of the first aspect of the present invention.

  In preferred embodiments, such food products are selected from the group consisting of fruits and fruit-derived products, vegetables and vegetable-derived products, cereals and cereal-derived products, dairy products, animal meat, poultry and marine products and mixtures thereof. The

  In a more preferred embodiment, the food product is a dairy product, preferably a medium temperature or thermophilic fermented dairy product, such as fresh cheese, yogurt, sour cream or tobalog.

  In another preferred embodiment, the food product is animal meat or chicken. In a preferred embodiment, said pharmaceutical product administers the antimicrobial composition of the first aspect of the invention to a human or animal to inhibit pathogenic microorganisms and alleviate symptoms associated with said pathogenic microorganisms. It is a useful product. Examples of such symptoms include symptoms associated with yeast infection. In such embodiments, the pharmaceutical product may be in unit dosage form containing an antimicrobial composition. Preferably, the unit dosage form may be a capsule or a tablet. However, the unit dosage form may be suitable for application to mucous membranes or skin, in which case it is in the form of a paste, cream, ointment or the like.

  Another aspect of the present invention relates to a method for producing a food, feed or pharmaceutical product according to the fifth aspect of the present invention. Adding an antimicrobial composition of this aspect.

In a preferred embodiment, the concentration of one or more Lactobacillus rhamnosus strains or Lactobacillus paracasei is greater than or equal to 1 × 10 ⁶ cfu / g, preferably for said food, feed or pharmaceutical product, 5x10 ⁶ cfu / g or more, most preferably 1x10 ⁸ cfu / g or more, or 1x10 ⁶ cfu / ml or more, preferably 5x10 ⁶ cfu / ml or more, most preferably 1x10 ⁸ cfu / ml or more, or food for feed or pharmaceutical product ^surface, 1x10 5 cfu / cm ² or more, preferably 5x10 ⁵ cfu / cm ² or more, and most preferably 1x10 ⁷ cfu / cm ² or more.

Where the food, feed or pharmaceutical product is produced by adding a composition containing one or more Lactobacillus rhamnosus strains and Lactobacillus paracasei, the one or more The concentration of Lactobacillus rhamnosus strain or Lactobacillus paracasei is 1 × 10 ⁶ cfu / g or more or 1 × 10 ⁶ cfu / ml or more with respect to the food, feed or pharmaceutical product, respectively. Or it is 1x10 < ⁵ > cfu / cm < ² > or more with respect to the surface of the said foodstuff, feed, or a pharmaceutical product. Preferably, the concentration of the one or more Lactobacillus rhamnosus strains or Lactobacillus paracasei is 5 × 10 ⁶ cfu / g or more, respectively, for the food, feed or pharmaceutical product, or 5 × 10 ⁶ cfu / ml or more, or 5 × 10 ⁵ cfu / cm ² or more with respect to the surface of the food, feed or pharmaceutical product. In a further aspect, the concentration of the one or more Lactobacillus rhamnosus strains or Lactobacillus paracasei is 1 × 10 ⁸ cfu / g for the food, feed or pharmaceutical product, respectively. Or 1 × 10 ⁸ cfu / ml or more, or 1 × 10 ⁷ cfu / cm ² or more with respect to the surface of the food, feed or pharmaceutical product.

  In a preferred embodiment, the production parameters are controlled so that the concentration of one or more Lactobacillus rhamnosus strains or Lactobacillus paracasei increases or remains constant during the production. Is done.

  The antimicrobial composition of the present invention is most conveniently used by being mixed with and / or applied to a mixable food, feed or pharmaceutical composition, but on the surface of a solid food product or its It is also effective to treat the interior of such products by injection. In still other embodiments, the composition may be applied as marinade, bread crumbs, seasoning rubs, nectar, colorant mixtures, etc., with important criteria being that the anti-microbial is degraded by bacteria as well as yeast. And available on mold-contaminated surfaces. In still other embodiments, the composition may be in contact with the food surface by applying the composition to a food container and applying the container to the food surface. The appropriate amount used may depend on the particular food product being treated and the method used to apply the composition to the food surface, but can be determined by simple experimentation.

  In a highly preferred embodiment, the method may comprise one or more fermentation steps, and the antimicrobial composition is the food, before, during or after such one or more fermentation steps. It may be added to feed or pharmaceutical products.

  In an even more preferred embodiment, the method comprises one or more strains of a genus selected from Lactobacillus, Streptococcus, Lactococcus and Leuconostoc, such as Lactobacillus bulgari. One or more strains of Lactobacillus bulgaricus and one or more strains of Streptococcus thermophilus, or one or more strains of Lactococcus lactis subsp. Lactis. , Leuconostoc mesenteroides subspecies cremolith ferment a milk substrate with a starting culture containing one or more strains of C. mesenteroides subsp. Cremoris and one or more strains of Lactococcus lactis subsp. diacetylactis Including that.

  The final aspect of the present invention is the Lactobacillus rhamnosus CHL C12697 or its mutant strain, Germanol of the Cultivation of Germulzrum Candum, which was deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM24616. Lactobacillus rhamnosus CHCC14226 or its mutant strain, German Collection of Microorganisms and Cell Cultures (DSM) deposited under the accession number DSM24652 Z) Lactobacillus paracasei CHCC12777 deposited under accession number DSM 24651 or a mutant thereof, wherein said mutant is obtained using said strain deposited as starting material .

  By using the strains deposited as starting material, those skilled in the art can use the known mutagenesis or reisolation techniques to retain the relevant features and advantages described herein. It will be apparent to those skilled in the art that mutations can be easily obtained. Thus, the term “its mutation” in the first aspect relates to a mutant strain obtained by using said strain deposited as starting material.

  Aspects of the invention are described below using non-limiting examples.

Example 1: Lb. Paracasei CHCC12777 and Lb. Semi-quantitative determination of the inhibitory effect of various yeasts and fungi on the use of Lb. rhamnosus CHCC12697 alone and in combination Lb. Paracasei CHCC12777 and Lb. For semi-quantitative testing of the inhibitory effect of Lb. rhamnosus CHCC12697 alone and in combination, an agar assay was used, which mimics the fresh cheese manufacturing process and product.

Full fat (3.5% w / w) homogenized milk was heat treated at 79 ± 1 ° C. for 20 minutes and quenched. A commercially available starting culture strain (Chr. Hansen A / S, F-DVS CHN-19 from Denmark) was seeded at 0.1 μl and the seeded milk was dispensed into 220 ml bottles. Lb. Paracasei CHCC12777 and / or Lb. Rb. Rhamnosus CHCC12697 was seeded at a total concentration of 5 × 10 ⁶ CFU / ml and one bottle served as a reference seeded with only the starting culture. In addition, 5% of the pH indicators bromcresol purple and bromcresol green were added to provide an indicator of the rate of acidification and to obtain a blue-green medium that can more easily detect the target yeast and mold. All bottles were incubated in a 29 ± 1 ° C. water bath and fermented under these conditions until the pH reached 4.60 ± 0.1. After fermentation, these bottles were quenched on ice and shaken vigorously to break up the coagulum. Then, the fermented milk was heated until the temperature reached 40 ° C., and 40 ml of 5% sterilized agar solution dissolved once and cooled to 60 ° C. was added. The fermented milk and agar solution was poured into a sterile petri dish and the plates were dried on a LAF bench for 30 minutes.

Selected yeasts and molds, Cry Vero My Kes-marxianus (Klyveromyces marxianus), Pichia Fell Men chest (Pichia fermentans), the Yarrowia lipolytica (Yarrowia lipolytica) and Candida sake (Candida sake), respectively ¹⁰ 4, Added dropwise at 10 ³ and 10 ² CFU / spot. Sufficiently germinated spore suspensions are diluted 1000, 100, 10 fold, Penicillium nargiovense Mucor ssp., Penicillium commune, and Cradiosporia sub The species (Cladiosporium ssp.) Was not diluted. The plates were incubated at 7 ± 1 ° C. and these were mainly tested for yeast and mold growth.

The results of the agar assay are shown in FIG. On agar plates (reference) made from milk fermented only with the starting culture, all tested yeasts and strains showed active growth. However, Lb. Paracasei CHCC12777 or Lb. When Lb. rhamnosus CHCC12697 was present in the course of milk fermentation, all concentrations of K. K. marxianus and Y.M. Lipolytica (Y. lipolytica) growth was inhibited. Furthermore, Lb. Paracasei CHCC12777 or Lb. When Rb. Rhamnosus CHCC12697 was present during the milk fermentation, significant inhibition was observed in all molds dripped onto the plates. Lb. Paracasei CHCC12777 and Lb. When any of the Rb. Rhamnosus CHCC12697 was added as a single strain, Fermentans or C. fermentans It did not cause an inhibitory effect on the salmon (C. sake) strain. However, Lb. Paracasei CHCC12777 and Lb. When milk was fermented in the presence of both Lb. rhamnosus CHCC12697, P. plutonium was dripped at a lower concentration of 10 ³ and 10 ² CFU / spot. Inhibition of growth of P. fermentans was observed, and Clariosporium ssp. And Mucor ssp. Paracasei CHCC12777 or Lb. Rb. Rhamnosus CHCC12697 inhibited growth more potently than when added alone. These results are shown in Lb. Paracasei CHCC12777 and Lb. Two strains of Lb. rhamnosus CHCC12697 have been shown to have a certain synergistic antifungal effect.

Example 2: Lb. Paracasei CHCC12777 and Lb. Semi-quantitative determination of the inhibitory effect of Lb. rhamnosus CHCC12697 alone and in combination on Yarrowia lipolytica Lb. Paracasei CHCC12777 and Lb. For semi-quantitative testing of the inhibitory effect of Lb. rhamnosus CHCC12697 alone and in combination, an agar assay was used, which mimics the fresh cheese manufacturing process and product.

Full fat (3.5% w / w) homogenized milk was heat treated at 79 ± 1 ° C. for 20 minutes and quenched. A commercially available starting culture strain (Chr. Hansen A / S, F-DVS CHN-19 manufactured by Denmark) was seeded at 0.1 μl, and the seeded milk was dispensed into 1 l bottles. Lb. Paracasei CHCC12777 and / or Lb. Rb. Rhamnosus CHCC12697 was seeded at a total concentration of 5 × 10 ⁶ CFU / ml and one bottle served as a reference seeded with only the starting culture. In addition, the two bottles include HOLDBAC ™ YM-B (50 DCU / 100 L; Danisco A / S, Denmark) or HOLDBAC ™ YM-C (20 DCU / 100 L; Danisco) with CHN-19 starting culture. A / S, Denmark). All bottles were incubated in a 29 ± 1 ° C. water bath and fermented under these conditions until the pH reached 4.60 ± 0.1. After fermentation, these bottles were quenched on ice and shaken vigorously to break up the coagulum.

The contents of each bottle were dispensed into smaller plastic cups and seeded with Yarrowia lipolytica isolated as a contaminant of fresh cheese products. In two separate cups with a volume of 1% (v / w), the final Y.V. It was seeded so that the contamination level in the fermented dairy product of Lipolytica was 1 × 10 ² CFU / g. The plastic cup was covered and stored at 8 ± 1 ° C. The fermented dairy product was plated on yeast glucose chloramphenicol (YGC) agar diluted 10-fold with physiological saline peptone and cultured at 25 ° C. for 5 days with aerobic incubation. The contamination level of Y. lipolytica was determined. Furthermore, the pH of the fermented milk material was measured throughout the storage period.

  As described in FIG. Paracasei CHCC12777 and Lb. When any of the Rb. Rhamnosus CHCC12697 was used as a single strain with the starting culture CHN-19 prior to fermentation, The growth of lipolytica (Y. lipolytica) was inhibited. Both strains caused more significant inhibition than HOLDBAC ™ YM-B and HOLDBAC ™ YM-C. Furthermore, Lb. Paracasei CHCC12777 and Lb. When Rb. Rhamnosus CHCC12697 was used in combination, a synergistic inhibitory effect was observed compared to when each strain was used alone. Lb. Paracasei CHCC12777 and Lb. Rb. Rhamnosus CHCC12697, when used in combination, is Y. in the fermented milk. It affects cell counts during both the induction and stationary phase of lipolytica, and this synergistic effect can contribute to extending shelf life of these types of products.

Example 3: Lb. Paracasei CHCC12777 and Lb. Semi-quantitative determination of the inhibitory effect of Lb. rhamnosus CHCC14226 alone and in combination on various yeast and mold contaminants Lb. Paracasei CHCC12777 and Lb. For semi-quantitative testing of the inhibitory effect of Lb. rhamnosus CHCC 14226 alone and in combination, an agar assay was used, which mimics the fresh cheese manufacturing process and product.

Full fat (3.5% w / w) homogenized milk was heat treated at 79 ± 1 ° C. for 20 minutes and quenched. A commercially available starting culture (F-DVS CHN-19) was seeded at 0.1 μl and the seeded milk was dispensed into 220 ml bottles. Lb. Paracasei CHCC12777 and / or Lb. Rb.rhamnosus CHCC14226 was seeded at a total concentration of 5 × 10 ⁶ CFU / ml and one bottle served as a reference seeded with only the starting culture. In addition, 5% of the pH indicators bromcresol purple and bromcresol green were added to provide an indicator of the rate of acidification and to obtain a blue-green medium that can more easily detect the target yeast and mold. All bottles were incubated in a 29 ± 1 ° C. water bath and fermented under these conditions until the pH reached 4.60 ± 0.1. After fermentation, these bottles were quenched on ice and shaken vigorously to break up the coagulum. Then, the fermented milk was heated until the temperature reached 40 ° C., and 40 ml of 5% sterilized agar solution dissolved once and cooled to 60 ° C. was added. The fermented milk and agar solution was poured into a sterile petri dish and the plates were dried on a LAF bench for 30 minutes.

Selected yeasts and molds, Cry Vero My Kes-marxianus (Klyveromyces marxianus), Pichia Fell Men chest (Pichia fermentans), the Yarrowia lipolytica (Yarrowia lipolytica) and Candida sake (Candida sake), respectively ¹⁰ 4, Added dropwise at 10 ³ and 10 ² CFU / spot. Sufficiently germinated spore suspensions are diluted 1000, 100, 10 fold, Penicillium nargiovense Mucor ssp., Penicillium commune, and Cradiosporia sub The species (Cladiosporium ssp.) Was not diluted. The plates were incubated at 7 ± 1 ° C. and these were mainly tested for yeast and mold growth.

The results of the agar assay are shown in FIG. On agar plates (reference) made from milk fermented only with the starting culture, all tested yeasts and strains showed active growth. However, Lb. When Lb. paracasei CHCC12777 was present during the milk fermentation, all concentrations of K. K. marxianus and Y.M. Lipolytica (Y. lipolytica) growth was inhibited. Lb. Rb. Rhamnosus CHCC 14226 also had all concentrations of K. pylori in the resulting plate. K. marxianus and the lowest to second Y. Lipolytica (Y. lipolytica) growth was inhibited. Lb. Paracasei CHCC12777 or Lb. Even when Lb. rhamnosus CHCC14226 was added as a single strain, they were found in low concentrations of P. aeruginosa. It showed a slight inhibitory effect on P. fermentans. However, Lb. Paracasei CHCC12777 and Lb. When Lb. rhamnosus CHCC 14226 is both present during the milk fermentation, P. cerevisiae added at the lowest concentration of 10 ² CFU / spot. The growth of P. fermentans was inhibited. In mold, Lb. Paracasei CHCC12777 or Lb. Significant inhibition was observed when Lb. rhamnosus CHCC14226 was present during the milk fermentation. Lb. Paracasei CHCC12777 and Lb. When the combination of Lb. rhamnosus CHCC14226 was used, the growth of Cladiosporium ssp. Paracasei CHCC12777 or Lb. Rb. Rhamnosus CHCC14226 was more potently inhibited compared to when it was added alone. These results are shown in Lb. Paracasei CHCC12777 and Lb. 2 shows that two strains of Rb. Rhamnosus CHCC14226 have a certain synergistic antifungal effect.

Example 4: Lb. Paracasei CHCC12777 and Lb. Semi-quantitative determination of the inhibitory effect of Lb. rhamnosus CHCC14226, alone and in combination, on Klyveromyces marxianus Lb. Paracasei CHCC12777 and Lb. For semi-quantitative testing of the inhibitory effect of Lb. rhamnosus CHCC 14226 alone and in combination, an agar assay was used, which mimics the fresh cheese manufacturing process and product.

Full fat (3.5% w / w) homogenized milk was heat treated at 79 ± 1 ° C. for 20 minutes and quenched. A commercially available starting culture strain (F-DVS CHN-19) was seeded at 0.1 μl and the seeded milk was dispensed into 1 l bottles. Lb. Paracasei CHCC12777 and / or Lb. Rb.rhamnosus CHCC14226 was seeded at a total concentration of 5 × 10 ⁶ CFU / ml and one bottle served as a reference seeded with only the starting culture. In addition, two bottles were seeded with HOLDBAC ™ YM-B (50 DCU / 100 L) or HOLDBAC ™ YM-C (20 DCU / 100 L) along with the CHN-19 starting culture. All bottles were incubated in a 29 ± 1 ° C. water bath and fermented under these conditions until the pH reached 4.60 ± 0.1. After fermentation, these bottles were quenched on ice and shaken vigorously to break up the coagulum.

The contents of each bottle were dispensed into smaller plastic cups and seeded with Klyveromyces marxianus isolated as a contaminant of fresh cheese products. In two separate cups, with a volume of 1% (v / w), the contamination level in the final fermented dairy product of Klyveromyces marxianus is 1 × 10 ² CFU / g Seeded. The plastic cup was covered and stored at 8 ± 1 ° C. The fermented dairy product is plated on yeast glucose chloramphenicol (YGC) agar diluted 10-fold with physiological saline peptone and cultured at 25 ° C. for 3-5 days with aerobic incubation. The contamination level of Velomyces marxianus was determined. Furthermore, the pH of the fermented milk material was measured throughout the storage period.

  As shown in FIG. The growth of K. marxianus was confirmed with Lb. Paracasei CHCC12777 and Lb. Rb. Rhamnosus CHCC14226 was inhibited when both were present and used as a single strain. As seen in Example 2, Lb. Parabasei CHCC12777 is significantly more potent than HOLDBAC ™ YM-B and HOLDBAC ™ YM-C. It caused inhibition of K. marxianus, but Lb. The inhibition caused by Lb. rhamnosus CHCC14226 was comparable to the HOLDBAC ™ culture. Lb. Paracasei CHCC12777 and Lb. When Rb. Rhamnosus CHCC14226 was used in combination, a coordinated inhibitory effect was observed compared to the inhibitory effect when each strain was used alone. When used in combination, Lb. Paracasei CHCC12777 and Lb. Rb. Rhamnosus CHCC 14226 is found in K. fermented milk. It can slow the growth of K. marxianus and contribute to extending the shelf life of this type of product.

Example 5: Lb. Paracasei CHCC 14676 and Lb. Semi-quantitative determination of the inhibitory effect of Lb. rhamnosus CHCC5366 alone and in combination on various mold contaminants Lb. Paracasei CHCC 14676 and Lb. For semi-quantitative testing of the inhibitory effects of Lb. rhamnosus CHCC5366 alone and in combination, an agar assay was used, which mimics the manufacturing process and product of yogurt.

Homogenized milk (1.5% w / w) was heat treated at 95 ° C. for 5 minutes and quenched. A commercially available starting culture (Chr. Hansen A / S, Denmark F-DVS YC-350) was seeded at 0.02%, and the seeded milk was dispensed into 220 ml bottles. Lb. Paracasei CHCC 14676, Lb. Rb. Rhamnosus CHCC5366, and combinations of the two, were seeded at a total concentration of 1 × 10 ⁷ CFU / ml. One bottle seeded with only the starting culture served as a reference. Furthermore, the pH indicators bromcresol purple and bromcresol green are 5% in all bottles to serve as an indicator of the rate of acidification and to obtain a blue-green medium that can more easily detect the target yeast and mold. Added. All bottles were incubated in a 43 ± 1 ° C. water bath and fermented under these conditions until the pH reached 4.60 ± 0.1. After fermentation, these bottles were quenched on ice and shaken vigorously to break up the coagulum. Then, the fermented milk was heated until the temperature reached 40 ° C., and 40 ml of 5% sterilized agar solution dissolved once and cooled to 60 ° C. was added. The fermented milk and agar solution was poured into a sterile petri dish and the plates were dried on a LAF bench for 30 minutes.

  Selected molds Penicillium nargiovese (1Ox), Penicillium commune (1Ox), Aspergillus versumol (1Ox) Of the completely germinated spore suspension was dropped onto the plate. These plates were incubated at 7 ° C. and tested for mold growth after an appropriate normal interval.

  The results of the agar assay are shown in FIG. All molds tested grew very well on agar plates made from milk fermented only with the starting culture (reference). However, Lb. Paracasei CHCC 14676 or Lb. Plates in which Rb. Rhamnosus CHCC5366 was present inhibited all mold growth. Furthermore, Lb. Paracasei CHCC 14676 and Lb. Penicillium commune, Aspergillus versicolor and Penicillium crustilus crustilus crusilis (Lb. rhhamnosus) CHCC5366 was dropped onto a plate that had been combined in the course of milk fermentation. ) Proliferation was markedly inhibited.

Example 6: Lb. Paracasei CHCC 14676 and Lb. Semi-quantitative determination of the inhibitory effect of Lb. rhamnosus CHCC14226 alone and in combination on various fungal contaminants Lb. Paracasei CHCC 14676 and Lb. For semi-quantitative testing of the inhibitory effect of Lb. rhamnosus CHCC14226 alone and in combination, an agar assay was used, which mimics the yogurt manufacturing process and product.

Homogenized milk (1.5% w / w) was heat treated at 95 ° C. for 5 minutes and quenched. A commercially available starting culture (Chr. Hansen A / S, Denmark F-DVS Yoflex® Mild) was seeded at 0.02%, and the seeded milk was dispensed into 220 ml bottles. Lb. Paracasei CHCC 14676, Lb. Rhamnosus CHCC 14226, and combinations of the two, were seeded at a total concentration of 1 × 10 ⁷ CFU / ml. One bottle seeded with only the starting culture served as a reference. Furthermore, the pH indicators bromcresol purple and bromcresol green are 5% in all bottles to serve as an indicator of the rate of acidification and to obtain a blue-green medium that can more easily detect the target yeast and mold. Added. All bottles were incubated in a 43 ± 1 ° C. water bath and fermented under these conditions until the pH reached 4.60 ± 0.1. After fermentation, these bottles were quenched on ice and shaken vigorously to break up the coagulum. Then, the fermented milk was heated until the temperature reached 40 ° C., and 40 ml of 5% sterilized agar solution dissolved once and cooled to 60 ° C. was added. The fermented milk and agar solution was poured into a sterile petri dish and the plates were dried on a LAF bench for 30 minutes.

  Selected molds Penicillium nargiovese (1Ox), Penicillium commune (1Ox), Aspergillus versumol (1Ox) Of the completely germinated spore suspension was dropped onto the plate. These plates were incubated at 7 ° C. and tested for mold growth after an appropriate normal interval.

  The results of the agar assay are shown in FIG. All molds tested grew very well on agar plates made from milk fermented only with the starting culture (reference). However, Lb. Paracasei CHCC 14676 or Lb. Plates in which Rb. Rhamnosus CHCC14226 was present inhibited the growth of all molds. Furthermore, Lb. Paracasei CHCC 14676 and Lb. Penicillium commune, Aspergillus versicolor and Penicillium crustilus crustilus crusilis (Lb. rhamnosus) CHCC14226 was dropped onto a plate that had been combined in the course of milk fermentation. ) Proliferation was markedly inhibited.

Example 7: Lb. Paracasei CHCC12777 and Lb. Semi-quantitative determination of the inhibitory effect of Lb. rhamnosus CHCC14226 alone and in combination on various fungal contaminants Lb. Paracasei CHCC12777 and Lb. For semi-quantitative testing of the inhibitory effect of Lb. rhamnosus CHCC14226 alone and in combination, an agar assay was used, which mimics the yogurt manufacturing process and product.

Homogenized milk (1.5% w / w) was heat treated at 95 ° C. for 5 minutes and quenched. A commercially available starting culture (Chr. Hansen A / S, Denmark F-DVS Yoflex® Mild) was seeded at 0.02%, and the seeded milk was dispensed into 220 ml bottles. Lb. Paracasei CHCC12777, Lb. Rhamnosus CHCC 14226, and combinations of the two, were seeded at a total concentration of 1 × 10 ⁷ CFU / ml. One bottle seeded with only the starting culture served as a reference. Furthermore, the pH indicators bromcresol purple and bromcresol green are 5% in all bottles to serve as an indicator of the rate of acidification and to obtain a blue-green medium that can more easily detect the target yeast and mold. Added. All bottles were incubated in a 43 ± 1 ° C. water bath and fermented under these conditions until the pH reached 4.60 ± 0.1. After fermentation, these bottles were quenched on ice and shaken vigorously to break up the coagulum. Then, the fermented milk was heated until the temperature reached 40 ° C., and 40 ml of 5% sterilized agar solution dissolved once and cooled to 60 ° C. was added. The fermented milk and agar solution was poured into a sterile petri dish and the plates were dried on a LAF bench for 30 minutes.

  Selected molds Penicillium nargiovese (1Ox), Penicillium commune (1Ox), Aspergillus versumol (1Ox) Of the completely germinated spore suspension was dropped onto the plate. These plates were incubated at 7 ° C. and tested for mold growth after an appropriate normal interval.

  The results of the agar assay are shown in FIG. All molds tested grew very well on agar plates made from milk fermented only with the starting culture (reference). However, Lb. Paracasei CHCC12777 or Lb. Plates in which Rb. Rhamnosus CHCC14226 was present inhibited the growth of all molds. Furthermore, Lb. Paracasei CHCC12777 and Lb. A significant inhibition was observed in the growth of all molds that were dripped onto the plates where Rb. Rhamnosus CHCC14226 was present in combination during the milk fermentation.

Lb. Challenge test with sour cream made with Lb. paracasei CHCC12777 Lb. P. commune, A. versicolor, P. brevicompactum, P. when using Lb. paracasei CHCC12777 as a single strain Sour cream was prepared to visually test the inhibitory effect on P. crustosum and P. glabrum.

Sterilized high fat milk was seeded at 0.01% with a commercially available heterofermentative starting culture (Chr. Hansen A / S, Denmark F-DVS DSG-2000). The milk was further seeded with OLDBAC ™ YM-B (10 DCU / 100 L) or Lactobacillus paracasei CHCC12777 (5 × 10 ⁶ CFU / g). One batch was seeded with only the starting culture as a reference.

  The milk was fermented at 28 ± 1 ° C. until the pH reached 4.60 ± 0.05 and the sour cream was post-treated. The sour cream was stirred and cooled to 20 ± 1 ° C. and stored at 7 ° C. ± 1 ° C.

  The day after the sour cream was prepared, various molds were sown on the surface of the sour cream so that there were 100 spores per spot on the surface of the sour cream. Mold growth was assessed visually after storage at 7 ° C. ± 1 ° C. for 45 days.

  The results of the sour cream test are shown in FIG. P. commune (M6), A. versicocolor (M7), P. brevicompactum (M1), P. crustosum ( M10) and P. glabrum (M8) are sour creams made from milk fermented with only the starting cell line (top), or the starting culture and the HOLDBAC ™ YM-B culture (middle) Above, it proliferated actively. In contrast, when Lactobacillus paracasei CHCC12777 was present during the milk fermentation, growth was inhibited in all of the molds tested.

Example 9: Challenge test on Tobalog made with Lactobacillus rhhamnosus CHCC12697 Mucor subspecies (mucor subspecies with Lactobacillus rhamnosus CHCC12697 as single strain Tovalogs were prepared to visually test their inhibitory effect on.

Sterilized low fat milk was seeded with 0.01% of a commercially available starting culture (Chr. Hansen A / S, Denmark F-DVS CHN-19). The milk was further seeded with OLDBAC ™ YM-B (5DCU / 100 L) or Lactobacillus rhamnosus CHCC12697 (5 × 10 ⁶ CFU / g). One batch was seeded with only the starting culture as a reference.

  The milk was fermented at 28 ± 1 ° C. until the pH reached 4.60 ± 0.05. The bathing temperature was 38-40 ° C. After aspiration, the card was pressurized with 1 bar for 30 minutes.

  The day after tobalog was prepared, mucor ssp. As a surface contaminant was seeded twice on the surface of tobalog (100 g) twice with a pipette so that there were 100 spores per spot. Mold growth was assessed visually after storage at 7 ° C. ± 1 ° C. for 18 days.

  The results of the tovalog test are shown in FIG. On tobalogs made from milk fermented using only the starting culture CHN-19, mucor ssp. Shows active growth. The fungus proliferates actively on tobalog made from milk cultured with the starting culture and the HOLDBAC ™ YM-B culture, although not as much as the starting culture and the HOLDBAC ™ YM-C culture. It also grew on tobalog made from milk cultured with the strain. However, when Lactobacillus rhamnosus CHCC12697 was present during the fermentation of milk, significant inhibition of mucor ssp. Growth was observed.

Example 10: Challenge test with yogurt made using Lactobacillus rhamnosus CHCC14226 P. brevicompactum using Lactobacillus rhamnosus CHCC14226 as a single strain 1.5% fat to visually test the inhibitory effect on P. communicum, P. commune, A. versicolor and P. crustosum. Of yogurt was prepared.

Homogenized milk (fat 1.5%) was heat treated at 95 ± 1 ° C. for 5 minutes in a 1 L bottle in a water bath and cooled rapidly. A commercial starting culture (Chr. Hansen A / S, F-DVS YF-L901 from Denmark) was seeded at 0.02%. The milk was further seeded with HOLDBAC ™ YM-B (20 DCU / 100 L) or Lactobacillus rhamnosus CHCC 14226 (1 × 10 ⁷ CFU / g). One bottle was seeded with only the starting culture as a reference.

  The milk was fermented at 43 ± 1 ° C. until the pH reached 4.60 ± 0.05. The formed yogurt was poured into a cup (100 g) and stored at 7 ± 1 ° C.

  The day after the yogurt was prepared, various molds were sown on the surface of the yogurt as surface contaminants in two yogurt cups so that there were 100 spores per spot. Mold growth was assessed visually after storage at 7 ° C. ± 1 ° C. for 45 days.

The result of the yogurt test is shown in FIG. P. brevicompactum (M1), P. commune (M6), Aspergillus versicolor (M7) P. crussum (M10) )
Shows active growth on yogurt made from milk fermented with only starting culture YF-L901 (upper) or starting culture and HOLDBAC ™ YM-B culture (middle) . In contrast, growth inhibition was observed in all molds tested when Lactobacillus rhamnosus CHCC14226 was present during the milk fermentation.

Example 11: K. in Tobalog Quantitative determination of the inhibitory effect of Lactobacillus paracasei CHCC12777 against K. lactis K. lactis using Lactobacillus paracasei CHCC12777 as a single strain. Tovalogs were prepared to quantitatively test the inhibitory effect on K. lactis.

Sterilized low fat milk was seeded with 0.01% of a commercially available starting culture (Chr. Hansen A / S, Denmark F-DVS CHN-19). The milk was further sown with OLDBAC ™ YM-B (20 DCU / 100 L) or Lactobacillus paracasei CHCC12777 (5 × 10 ⁶ CFU / g). One batch was seeded with only the starting culture as a reference.

  The milk was fermented at 28 ± 1 ° C. until the pH reached 4.60 ± 0.05. The bathing temperature was 38-40 ° C. After aspiration, the card was pressurized with 1 bar for 30 minutes.

  The next day after the tovalog was prepared, approximately 10 g of tobalog sample blocks were cut out and placed in a stomacher bag. The block was inoculated twice with 10.000 CFU / ml of yeast inside using a thin needle. The bag covered the tobalog and was sealed with tape.

  The samples are stored at 7 ± 1 ° C. The level of K. lactis contamination was analyzed. Analysis was performed by plating 1 g of Tovalog and an appropriate 1-fold dilution of sarinpeptone on Yeast Glucose Chloramphenicol (YGC) agar and incubating it aerobically at 25 ° C. for 5 days. .

  As shown in FIG. 11, in Tovalog where Lactobacillus paracasei CHCC12777 was present together with the starting culture CHN-19 before fermentation, The growth of Lactis was inhibited. Lactobacillus paracasei CHCC12777 caused a significantly higher inhibition than the commercial culture HOLDBAC ™ YM-B.

Example 12: K. in Tobalog Quantitative determination of the inhibitory effect of Lactobacillus rhamnosus CHCC12697 on Lactis (K. lactis) When K. lactis is used as a single strain, L. bacillus rhamnosus. Tovalogs were prepared to quantitatively test the inhibitory effect on K. lactis.

Sterilized low fat milk was seeded with 0.01% of a commercially available starting culture (Chr. Hansen A / S, Denmark F-DVS CHN-19). The milk was further seeded with OLDBAC ™ YM-B (5DCU / 100 L) or Lactobacillus rhamnosus CHCC12697 (5 × 10 ⁶ CFU / g). One batch was seeded with only the starting culture as a reference.

  The milk was fermented at 28 ± 1 ° C. until the pH reached 4.60 ± 0.05. The bathing temperature was 38-40 ° C. After aspiration, the card was pressurized with 1 bar for 30 minutes.

  The next day after the tovalog was prepared, approximately 10 g of tobalog sample blocks were cut out and placed in a stomacher bag. The block was inoculated twice with 10.000 CFU / ml of yeast inside using a thin needle. The bag covered the tobalog and was sealed with tape.

  The samples are stored at 7 ± 1 ° C. The level of K. lactis contamination was analyzed. Analysis is performed by plating 1 g of Tovalog and an appropriate 1-fold dilution in sarin peptone on Yeast Glucose Chloramphenicol (YGC) agar and incubating it aerobically at 25 ° C. for 5 days. It was.

  As shown in FIG. 11, in Tobalog where Lactobacillus rhamnosus CHCC12697 was present with the starting culture CHN-19 prior to fermentation, The growth of Lactis was inhibited. Lactobacillus rhamnosus CHCC12697 caused a significantly higher inhibition than the commercial culture HOLDBAC ™ YM-B.

Example 13: Quantitative determination of the inhibitory effect of Lactobacillus rhamnosus CHCC14226 against Debaromyces hansenii in yogurt using Lactobacillus 26 as a single strain Lactobacillus 26 as Lactobacillus 26 Yogurt was prepared to quantitatively test its inhibitory effect on Debaromyces hansenii.

Homogenized milk (1.5% w / w) was heat treated at 95 ° C. for 5 minutes in a water bath in a 1 L bottle and quenched. A commercially available starting culture (Chr. Hansen A / S, Denmark F-DVS YF-L901) was seeded at 0.02%. The milk was further seeded with HOLDBAC ™ YM-B (20 DCU / 100 L) or Lactobacillus rhamnosus CHCC 14226 (1 × 10 ⁷ CFU / ml). One bottle seeded with only the starting culture served as a reference.

  The milk was fermented at 43 ° C. ± 1 ° C. until the pH reached 4.60 ± 0.1. The prepared yogurt was poured into a cup (100 g) and stored at 7 ° C. ± 1 ° C.

  The day after the yogurt was prepared, 1 ml of yeast was inoculated per cup so as to be 20 CFU / g in two yogurt cups. The yeast was uniformly dispersed in yogurt. The cup is capped, stored at 7 ° C., and D.I. Contamination of D. hansenii was analyzed. The analysis was performed by plating 1 g yogurt and an appropriate 1-fold dilution of sarinpeptone on Yeast Glucose Chloramphenicol (YGC) agar and incubating it aerobically at 25 ° C. for 5 days. .

  As shown in FIG. Growth of D. hansenii was inhibited when Lactobacillus rhamnosus CHCC14226 was present with the starting culture YF-L901 prior to fermentation. The strain caused a significantly higher inhibition than the commercial culture HOLDBAC ™ YM-B.

Deposits and solutions of those skilled in the art Applicants require that the following deposited samples of microorganisms must be available to those skilled in the art before the patent is subject to registration assessment.

  Lactobacillus rhamnosus CHCC12697 was published on March 1, 2011, in Microorganisms and Cell Cultures (Deutsche Sammlung von Mikhlogenismanund Zellkultur. 7B, deposited with D-38124 Braunschweig, with accession number DSM24616.

  Lactobacillus rhamnosus CHCC 14226, on March 15, 2011, Microorganisms and Cell Cultures (Deutsche Sammlung von Mikhlogenis Zundkulh); 7B, D-38124 Braunschweig, deposited with accession number DSM24652.

  Lactobacillus paracasei CHCC12777 was published on March 15, 2011, in Microorganisms and Cell Cultures (Deutsche Sammlung von Mikhlogensund und Zellkultur. 7B, D-38124 Braunschweig, deposited with accession number DSM 24651.

  These deposits were made in accordance with the Budapest Treaty on the International Approval of the Deposit of Microorganisms for Patent Procedures.

Reference US 2005/0095318 (Schwenninger et al.)
WO 2004/041305 (Vario Ltd)
Tharmaraj, N.A. & Shah, N .; P. (2009) Antimicrobial effects of probabilistic bacteria against selected species of molds in cheese-based dips. International Journal of Food Science & Technology. 44: 1916-1926.

Claims (13)

One or more of Lactobacillus rhamnosus strains and Lactobacillus paracasei strains, wherein the one or more Lactobacillus rhamnosus R. Ractobacillus rhamnosus CHCC12697, and Lactobacillus rhamnosus CHCC14226 with accession number DSM24652, selected from the group consisting of Lactobacillus paracazei (Lactobacillus sp. Rakazei (Lactobacillus paracasei) is CHCC12777, antimicrobial composition. The following groups:
Lactobacillus rhamnosus CHCC12697, deposited with German Collection of Microorganisms and Cell Cultures (DSMZ) with accession number DSM24616,
German Collection of Microorganisms and Cell Cultures (DSMZ), deposited with the accession number DSM24652, Lactobacillus rhamnosus Cul 14 with the Cact. Lactobacillus paracasei CHCC12777;
Containing one or more strains selected from the antimicrobial composition. Use of the antimicrobial composition according to claim 1 or 2 in the preparation of food, feed or pharmaceutical products. Use of the antimicrobial composition according to claim 1 or 2 as a pharmaceutical product. A food, feed or pharmaceutical product comprising the antimicrobial composition according to claim 1 or 2 . 6. The food product of claim 5 , selected from the group consisting of fruits and fruit-derived products, vegetables and vegetable-derived products, cereals and cereal-derived products, dairy products, animal meat, poultry and marine products and mixtures thereof. 6. A pharmaceutical product according to claim 5 , which is a unit dosage form containing the composition according to claim 1 or 2 . A method for producing the food, feed or pharmaceutical product according to any one of claims 5 to 7 , wherein the antimicrobial composition according to claim 1 or 2 is the food, feed or pharmaceutical product. The said manufacturing method including the process added to. It is a manufacturing method of Claim 8 , Comprising:
(A) in the process of producing the food, feed or pharmaceutical product, the antimicrobial composition according to any of claims 1 or 2 is applied to one or more Lactobacillus rhamnosus strains or one or more Lactobacillus paracasei strain of 1 × 10 ⁶ cfu / g or more or 1 × 10 ⁶ cfu / ml or more to the food, feed or pharmaceutical product, or 1 × 10 ⁵ cfu on the surface of the food, feed or pharmaceutical product It added to a concentration of / cm ² steps, and (b) one or more Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain or concentration of one or more of Lactobacillus paracasei (Lactobacillus paracasei) strain Large and, or so that a constant, comprising the step of controlling the manufacturing parameters during the course of the production, the production method. 10. A method according to any of claims 8 or 9 , comprising one or more fermentation steps.   Lactobacillus rhamnosus strain CHCC12697, deposited with German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM24616.   Lactobacillus rhamnosus strain CHCC14226 deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM24652.   Lactobacillus paracasei CHCC12777 strain deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under the accession number DSM24651.

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